Utilization of Nanoparticles for Photoacoustic Chemical Imaging

Tumors are known to have unique chemical properties, such as low pH (acidosis), high K+ (hyperkalemia), and low O2 (hypoxia). Tumor acidosis has been known to influence therapeutic activities of chemotherapeutic drugs. Another conventional cancer treatment, radiation therapy, is highly dependent on local oxygen concentrations. Hyperkalemia has been recently reported to suppress the immune response of activated T-cells. It is also believed that the spatial distribution of these analytes and its heterogeneity, are of relevance. Despite the importance of such chemical information on tumors, there are no clinically available tools for “quantitative” pH, K+, or tissue O2 imaging. Here, photoacoustic (PA) imaging is employed to provide chemical imaging of all these target analytes for cancer (pH, O2 and K+). As for pH, we report on an in vivo pH mapping nanotechnology. This subsurface chemical imaging is based on tumor-targeted, pH sensing nanoprobes and multi-wavelength photoacoustic imaging (PAI). The nanotechnology consists of an optical pH indicator, SNARF-5F, 5-(and-6)-Carboxylic Acid, encapsulated into polyacrylamide nanoparticles with surface modification for tumor targeting. Facilitated by multi-wavelength PAI plus a spectral unmixing technique, the accuracy of pH measurement inside the biological environment is not susceptible to the background optical absorption of biomolecules, i.e., hemoglobins. As a result, both the pH levels and the hemodynamic properties across the entire tumor can be quantitatively evaluated with high sensitivity and high spatial resolution in in vivo cancer models. For K+, we extend this technique to ion-selective photoacoustic optodes (ISPAOs) that serve at the same time as fluorescence-based ISOs, and apply it specifically to potassium (K+). However, unfortunately, sensors capable of providing potassium images in vivo are still a future proposition. Here, we prepared an ion-selective potassium nanosensor (NS) aimed at in vivo photoacoustic (PA) chemical imaging of the extracellular environment, while being also capable of fluorescence based intracellular ion-selective imaging. This potassium nanosensor (K+ NS) modulates its optical properties (absorbance and fluorescence) according to the potassium concentration. The K+ NS is capable of measuring potassium, in the range of 1 mM to 100 mM, with high sensitivity and selectivity, by ISPAO based measurements. Also, a near infrared dye surface modified K+ NS allows fluorescence-based potassium sensing in the range of 20 mM to 1 M. The K+ NS serves thus as both PA and fluorescence based nanosensor, with response across the biologically relevant K+ concentrations, from the extracellular 5 mM typical values (through PA imaging) to the intracellular 150 mM typical values (through fluorescence imaging). Lastly, nano-enabled tissue O2 monitoring by PA, called lifetime-based PA (PALT) imaging, was introduced and demonstrated. A known PALT oxygen indicator, Oxyphor G2, is conjugated into polyacrylamide nanoparticles, called G2-PAA NP. The oxygen sensing capability of the G2-PAA NP has been confirmed in vitro and in vivo studies. In an Appendix, we show how to monitor photodynamic therapy (PDT) using the PALT approach to measure the local oxygen depletion as a function of PDT time. Oxygen depletion during PDT is monitored using both oximeter and PALT spectroscopy in vitro. The latter is enabled by theranostic NPs of methylene blue (MB) conjugated PAA, used for both PALT and PDT. This synergistic approach has good potential for personalized medicine.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/143924/1/lechang_1.pd

Quantification of Proteins and Cells: Luminometric Nonspecific Particle-Based Methods

New luminometric particle-based methods were developed to quantify protein and to count cells. The developed methods rely on the interaction of the sample with nano- or microparticles and different principles of detection. In fluorescence quenching, timeresolved luminescence resonance energy transfer (TR-LRET), and two-photon excitation fluorescence (TPX) methods, the sample prevents the adsorption of labeled protein to the particles. Depending on the system, the addition of the analyte increases or decreases the luminescence. In the dissociation method, the adsorbed protein protects the Eu(III) chelate on the surface of the particles from dissociation at a low pH. The experimental setups are user-friendly and rapid and do not require hazardous test compounds and elevated temperatures. The sensitivity of the quantification of protein (from 40 to 500 pg bovine serum albumin in a sample) was 20-500-fold better than in most sensitive commercial methods. The quenching method exhibited low protein-to-protein variability and the dissociation method insensitivity to the assay contaminants commonly found in biological samples. Less than ten eukaryotic cells were detected and quantified with all the developed methods under optimized assay conditions. Furthermore, two applications, the method for detection of the aggregation of protein and the cell viability test, were developed by utilizing the TR-LRET method. The detection of the aggregation of protein was allowed at a more than 10,000 times lower concentration, 30 μg/L, compared to the known methods of UV240 absorbance and dynamic light scattering. The TR-LRET method was combined with a nucleic acid assay with cell-impermeable dye to measure the percentage of dead cells in a single tube test with cell counts below 1000 cells/tube.Siirretty Doriast

Definition of a near real time microbiological monitor for space vehicles

Efforts to identify the ideal candidate to serve as the biological monitor on the space station Freedom are discussed. The literature review, the evaluation scheme, descriptions of candidate monitors, experimental studies, test beds, and culture techniques are discussed. Particular attention is given to descriptions of five candidate monitors or monitoring techniques: laser light scattering, primary fluorescence, secondary fluorescence, the volatile product detector, and the surface acoustic wave detector

Aplicação da citometria de fluxo ao estudo do genoma vegetal

Doutoramento em BiologiaHoje em dia, a aplicação da citometria de fluxo (CMF) no estudo do genoma vegetal consiste maioritariamente em análises do nível de ploidia e em estimativas do conteúdo em ADN nuclear. Esta Tese de Doutoramento tem como objectivo aplicar a CMF e outras técnicas citológicas, como a microscopia de fluorescência e a hibridização in situ, no estudo do genoma vegetal. O primeiro Capítulo introduz a CMF, nomeadamente o seu funcionamento, aspectos metodológicos da análise do conteúdo em ADN nuclear em plantas, e aplicações da CMF na biologia vegetal. No Capítulo II são apresentados dois estudos sobre a estabilidade do nível de ploidia durante o processo de cultura in vitro de sobreiro (Capítulo II.1) e zimbro (Capítulo II.2), duas espécies lenhosas de elevada importância económica e ecológica. No Capítulo III são apresentados os resultados das análises de conteúdo em ADN nuclear efectuadas em três grupos de espécies vegetais. No Capítulo III.1 a CMF foi utilizada para estimar pela primeira vez o tamanho do genoma nuclear de cultivares de oliveira e zambujeiro; a variação intraspecífica como tópico de elevado interesse é aqui discutida tendo em consideração as boas práticas. No Capítulo III.2 foi analisado o conteúdo em ADN nuclear de três espécies de Ulmaceae da Península Ibérica que contêm compostos mucilaginosos, conhecidos por interferirem com as análises por CMF; é também apresentado um protocolo eficiente que previne o efeito negativo deste compostos e permite uma correcta análise do tamanho do genoma de espécies com problemas similares. No Capítulo III.3 é apresentado um estudo extensivo de CMF e citogenética molecular realizado em espécies de festuca da Península Ibérica. Considerando os resultados obtidos nos Capítulos II e III, no Capítulo IV foram efectuados estudos inovadores de índole metodológica. No Capítulo IV.1 é apresentada a primeira comparação sistemática de tampões de isolamento nuclear, enquanto no Capítulo IV.2 é estudado o efeito negativo do ácido tânico, um composto fenólico conhecido por interferir com os núcleos vegetais e com as estimativas de conteúdo em ADN. De acordo com os resultados obtidos nestes estudos, no Capítulo IV.3 foram testados dois novos tampões de lise nuclear em 37 espécies vegetais. No Capítulo V é introduzida a base de dados “Plant DNA Flow Cytometry (FLOWER)”. Esta base de dados contém informações retiradas de mais de 700 publicações na área da CMF vegetal, disponibilizando-as de uma forma atraente e permitindo a realização de análises quantitativas respeitantes a muitos tópicos importantes. Finalmente, no Capítulo VI são apresentadas as conclusões da presente Tese de Doutoramento, com especial incidência nas boas práticas necessárias para estimar o conteúdo em ADN nuclear em plantas e nas direcções futuras da CMF no estudo do genoma vegetal.Nowadays, the application of flow cytometry (FCM) to study plant genomes is mostly focused on DNA ploidy level analyses and nuclear DNA content estimations. The objective of this PhD Thesis is to applly FCM and related techniques, as fluorescence microscopy and in situ hybridization, to study plant genomes. The first chapter introduces FCM, namely the functioning of this technique, the methodological aspects of the analysis of nuclear DNA content in plants, and the applications of FCM in plant sciences. In Chapter II, studies of ploidy stability of the in vitro culture process of two economically important woody plant species, Quercus suber (Chapter II.1) and Juniperus phoenicea (Chapter II.2), are presented. In Chapter III, three studies regarding nuclear DNA content analyses in plant species are given. In Chapter III.1 the genome size of olive cultivars and wild olive was estimated for the first time using FCM; the hot topic of intraspecific variation is discussed here with respect to best practices. In Chapter III.2, the nuclear DNA content of three Ulmaceae species, containing mucilaginous compounds that interfere with FCM analysis, is analysed; an efficient protocol that circumvented this problem and enabled reliable genome size estimations in these species is presented. A through flow cytometric and molecular cytogenetic study on fescue species of the Iberian Peninsula is performed in Chapter III.3. In light with the results obtained in Chapters II and III, innovative methodological studies were performed in Chapter IV. In Chapter IV.1 the first systematic comparison of lysis buffers is presented, while in Chapter IV.2 the negative effect of tannic acid, a common phenolic compound known to interfere with plant nuclei and DNA content estimations, is analysed. According with the results obtained in these studies, in Chapter IV.3 two new lysis buffers were tested with a set of 37 plant species. In chapter V the Plant DNA Flow Cytometry Database (FLOWER) is introduced. The FLOWER database collects information from more than 700 publications in this area of FCM, and makes it accessible in one user-friendly design that enables quantitative analysis of many important topics. Finally in Chapter VI the conclusions of the present PhD Thesis are presented with special focus on a compilation of best practices for nuclear DNA estimation using FCM and the future directions on the use of this technique to study plant genomes

CMOS system for high throughput fluorescence lifetime sensing using time correlated single photon counting

Fluorescence lifetime sensing using time correlated single photon counting (TCSPC) is a key analytical tool for molecular and cell biology research, medical diagnosis and pharmacological development. However, commercially available TCSPC equipment is bulky, expensive and power hungry, typically requiring iterative software post-processing to calculate the fluorescence lifetime. Furthermore, the technique is restrictively slow due to a low photon throughput limit which is necessary to avoid distortions caused by TCSPC pile-up. An investigation into CMOS compatible multimodule architectures to miniaturise the standard TCSPC set up, allow an increase in photon throughput by overcoming the TCSPC pile-up limit, and provide fluorescence lifetime calculations in real-time is presented. The investigation verifies the operation of the architectures and leads to the selection of optimal parameters for the number of detectors and timing channels required to overcome the TCSPC pile-up limit by at least an order of magnitude. The parameters are used to implement a low power miniaturised sensor in a 130 nm CMOS process, combining single photon detection, multiple channel timing and embedded pre-processing of the fluorescence lifetime, all within a silicon area of < 2 mm2. Single photon detection is achieved using an array of single photon avalanche diodes (SPADs) arranged in a digital silicon photomultiplier (SiPM) architecture with a 10 % fill-factor and a compressed 250 ps output pulse, which provides a photon throughput of > 700 MHz. An array of time-interleaved time-to-digital converters (TI-TDCs) with 50 ps resolution and no processing dead-time records up to eight photon events during each excitation period, significantly reducing the effect of TCSPC pile-up. The TCSPC data is then processed using an embedded centre-of-mass method (CMM) pre-calculation to produce single exponential fluorescence lifetime estimations in real-time. The combination of high photon throughput and real-time calculation enables advances in applications such as fluorescence lifetime imaging microscopy (FLIM) and time domain fluorescence lifetime activated cell sorting. To demonstrate this, the device is validated in practical bulk sample fluorescence lifetime, FLIM and simulated flow based experiments. Photon throughputs in excess of the excitation frequency are demonstrated for a range of organic and inorganic fluorophores for minimal error in lifetime calculation by CMM (< 5 %)

Novel Technologies for Real-Time Fluorescent Lifetime Imagind Data Acquisition and Processing for Clinical Diagnosis

Endogenous Fluorescence Lifetime Imaging (FLIM) is a noninvasive technique that has been explored with promising results in a wide range of biomedical applications, including clinical diagnosis. A central issue for the translation of FLIM into the medical field is the development of a robust, fast and cost-effective FLIM instrumentation suitable for in vivo tissue imaging. This thesis directly addressed some of the technical limitations that must be overcome to enable clinical applications of FLIM. The following specific aims were accomplished. First, endogenous FLIM imaging and high-resolution reflectance confocal microscopy (RCM) were integrated into a multimodal bench-top optical system. This multimodal system was used to image oral epithelial cancer in a hamster cheek pouch model. Second, an endoscopic system for fast (0.5-4 frames/second) endogenous wide-field FLIM imaging of oral lesions was developed. The FLIM endoscope system is being evaluate at Texas A&M University College of Dentistry, where more than 80 patients presenting oral lesions suspected of pre-cancer or cancer have been imaged up to date. Third, a novel fluorescence lifetime estimation algorithm was developed to achieve robust, accurate, and real-time fluorescence lifetime estimation. This algorithm is enabling real-time FLIM image processing and visualization during the endoscopic examination of patients with suspicious oral lesions. Finally, the endoscopic endogenous FLIM data from suspicious oral lesions collected at the Texas A&M College of Dentistry was used to develop machine learning algorithms for automated identification of precancerous and cancerous lesions from benign oral epithelial lesions. Our results indicate that endogenous FLIM endoscopy can detect oral epithelial pre-cancer and cancer from a wider range of benign conditions, with levels of sensitivity and specificity above 85%. Altogether, this work has demonstrated the potentials of endogenous FLIM endoscopy as a clinical tool for early detection of oral epithelial cancer

A compendium of single extracellular vesicle flow cytometry

Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV-containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt-EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses

Circadian patterns in key physiological processes of the marine diatom Phaeodactylum tricornutum

Circadian rhythms are a widespread feature among all the levels of organismal complexity, from bacteria to humans. Nevertheless their ecological relevance in marine phytoplankton has not been clearly assessed. In this study the diatom P. tricomutum, was used as a model for giving an integrated picture of the circadian patterns of phytoplankton photophysiology in relation to the external light field and for assessing the interaction between the cell cycle and the temporal control of processes like pigment synthesis and photosynthesis. The results suggest that light/dark shifts are crucial for regulating the timing of different cell activities, including the synthesis of photoprotectant pigments (i.e., diadinoxanthin). The photosynthetic processes run at different paces in different stages of the cell cycle and different times of the day. This would imply a synchronization of cell processes due to both internal and external entrainer/s. When the external trigger is removed (e.g., in absence of light/dark shifts), the time distribution of the cell cycle stages quickly flattens, the efficiency of the use of light lowers, as well as the flexibility in facing light variations. Though, this does not always involve the loss of synchronization of processes like pigment synthesis, or the activation/inactivation of the photosynthetic machinery. The results from an in situ experiment support the occurrence of a circadian regulation of photophysiological processes in natural phytoplankton, at least in the upper layer of the water column, where solar radiation can act as a signal for the entrainment of circadian rhythms, whether or not mediated by specific photoreceptors. The overall picture is that the presence of systems for the circadian control of physiological activity would be a successful feature in marine phytoplankton, which is reasonable also in an evolutionary perspective, since the day/night cycle is a permanent pattern in the Earth system